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Abstract: Although highly efficient, modification of a genomic site by the CRISPR enzyme requires the generation of a sgRNA unique to the target site(s) beforehand. This work describes the key steps leading to the construction of efficient sgRNA vectors using a strategy that allows the efficient detection of the positive colonies by PCR prior to DNA sequencing. Since efficient genome editing using the CRISPR system requires a highly efficient sgRNA, a preselection of candidate sgRNA targets is necessary to save time and effort. A dual luciferase reporter system has been developed to evaluate knockout efficiency by examining double-strand break repair via single strand annealing. Here, we use this reporter system to pick up the preferred xCas9/sgRNA target from candidate sgRNA vectors for specific gene editing. The protocol outlined will provide a preferred sgRNA/CRISPR enzyme vector in 10 days (starting with appropriately designed oligonucleotides).
Fuente: J Vis Exp . 2020 Dec 5;(166).
Editorial: MYJoVE Corporation
Año de publicación: 2020
Nº de páginas: 15
Tipo de publicación: Artículo de Revista
DOI: 10.3791/59639
ISSN: 1940-087X
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LI, HEGANG
QIN, HUAIYUAN
ZHANG, NING
ZHAO, JINSHAN
XIN, JINGJING
FLOR MARIA PEREZ CAMPO
LIU, HUAWEI
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